DOCSLIB.ORG

Novel Sources of Β-Glucanase for the Enzymatic Degradation Of

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Novel Sources of Β-Glucanase for the Enzymatic Degradation Of Enzyme and Microbial Technology 52 (2013) 203–210 Contents lists available at SciVerse ScienceDirect Enzyme and Microbial Technology jou rnal homepage: www.elsevier.com/locate/emt ␤ ଝ Novel sources of -glucanase for the enzymatic degradation of schizophyllan a a,∗ a a Nongnuch Sutivisedsak , Timothy D. Leathers , Kenneth M. Bischoff , Melinda S. Nunnally , b Stephen W. Peterson a Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture 1815 North University Street, Peoria, IL 61604, USA b Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture 1815 North University Street, Peoria, IL 61604, USA a r t i c l e i n f o a b s t r a c t Article history: Schizophyllan is a homoglucan produced by the fungus Schizophyllum commune, with a ␤-1,3-linked Received 29 October 2012 ␤ backbone and -1,6-linked side chains of single glucose units at every other residue. Schizophyllan is Received in revised form 3 December 2012 commercially produced for pharmaceutical and cosmetics uses. However, surprisingly little information Accepted 4 December 2012 is available on the biodegradation of schizophyllan. Enzymes that attack schizophyllan could be useful for controlled modifications of the polymer for novel applications. Enrichment cultures were used to Keywords: isolate 20 novel fungal strains from soil samples, capable of growing on schizophyllan as a sole carbon ␤-glucanase source. Three additional strains were isolated as contaminants of stored schizophyllan solutions. Strains Hypocrea nigricans ␤ showing the highest levels of -glucanase activity were identified as Penicillium simplicissimum, Penicil- Penicillium simplicissimum lium crustosum, and Hypocrea nigricans. ␤-glucanases also showed activity against the similar ␤-glucans, Penicillium crustosum ␤ Schizophyllum commune laminarin and curdlan. By comparison, commercial -glucanase from Trichoderma longibrachiatum and Schizophyllan laminarinase from Trichoderma sp. showed lower specific activities toward schizophyllan than most of ␤ the novel isolates. -glucanases from P. simplicissimum and H. nigricans exhibited temperature optima of ◦ ◦ 60 C and 50 C against schizophyllan, respectively, with broad pH optima around pH 5.0. Partial purifi- ␤ cations of -glucanase from P. simplicissimum and P. crustosum demonstrated the presence of multiple active endoglucanase species, including a 20–25 kD enzyme from P. simplicissimum. Published by Elsevier Inc. 1. Introduction this assumption. However, surprisingly little information is avail- able on the biodegradation of schizophyllan. S. commune has been Schizophyllan is a polysaccharide produced by Schizophyllum reported to produce endo-␤-1,3-glucanase [5], and Rau [2] pro- commune, a white-rot fungus and ubiquitous mushroom. It is a posed that the organism can consume schizophyllan as a carbon ␤ ␤ homoglucan with a -1,3-linked backbone and single -1,6-linked source, contributing to a loss of polysaccharide molecular weight glucose side chains at every other residue [1,2]. Schizophyllan in late cultures. Lo et al. [6] described ␤-glucosidases from S. com- acts as a biological response modifier and a non-specific stimu- mune. Fontaine et al. [7] reported that schizophyllan was slightly lator of the immune system. It is used in vaccines, anti-cancer hydrolyzed by one of two exo-␤-1,3-glucanases associated with the therapies, and as a bioactive cosmetics ingredient. Schizophyl- cell walls of Aspergillus fumigatus. On the other hand, Kanzawa et al. lan can form oxygen-impermeable films for food preservation [3]. [8] found that exo-␤-1,3-glucanase from Bacillus circulans rapidly It also has been tested for use in enhanced petroleum recovery hydrolyzed curdlan and laminarin, but did not attack schizo- [2,4]. phyllan. Tanji et al. [9] reported that schizophyllan was partially As a natural polysaccharide, it can be assumed that schizo- degraded at a very slow rate in rats, to lower molecular weight phyllan is biodegradable, and many of its applications rely on forms of <10,000 that were excreted in urine. It is potentially valu- able to identify enzymes that attack schizophyllan, particularly for use in controlled modifications of the polymer for novel applica- tions. In the current study, 23 novel strains were isolated that were ଝ Mention of any trade names or commercial products in this publication is solely capable of growing on schizophyllan as a sole carbon source. Strains for the purpose of providing specific information and does not imply recommen- showing the highest activities against schizophyllan were identi- dation or endorsement by the U.S. Department of Agriculture. USDA is an equal fied, and ␤-glucanase activities were characterized. Results indicate opportunity provider and employer. ∗ that novel fungal isolates are promising sources of schizophyllan- Corresponding author. Tel.: +1 309 681 6620; fax: +1 309 681 6040. E-mail address: [email protected] (T.D. Leathers). degrading enzymes. 0141-0229/$ – see front matter. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.enzmictec.2012.12.002 204 N. Sutivisedsak et al. / Enzyme and Microbial Technology 52 (2013) 203–210 Table 1 containing 1.0% (w/v) commercial schizophyllan in a 50 mL flask with three 10 mm a ◦ ␤-glucanase activity against schizophyllan produced by newly isolated strains glass beads. Cultures were incubated at 200 rpm for 7 days at 28 C, then centrifuged × ␤ at 3220 g to produce cell-free culture supernatants. Mycelial pellets were dried at Strain number Isolation sites -glucanase ◦ b 60 C for 48 h. All experiments were carried out in triplicate and standard deviations (near Peoria, Illinois) (U activity/mL) are shown. 1-1 Woodland 0.24 ± 0.02 1-2 0.16 ± 0.03 1-3 0.17 ± 0.03 2.4. Enzyme and protein assays ± 2-1 Prairie grassland 0.005 <0.001 Quantitative ␤-glucanase assays were performed by the dinitrosalicylic acid 2-2 <0.001 (DNS) method [14] as modified by Leathers et al. [15]. Samples (5–20 ␮L) were 2-3 0.003 ± <0.001 ␮ incubated in a total volume of 205 L containing 0.5% (w/v) substrate (schizo- 2-4 0.001 ± <0.001 ␤ ◦ phyllan or another -glucan) in 50 mM sodium acetate buffer, pH 5.0, at 28 C. 3-1 Hayfield/woodland <0.001 Sample dilutions and incubation times were adjusted to ensure results were within 3-2 border <0.001 the linear range of the assays. One unit of enzyme activity is defined as the amount of enzyme necessary to release 1 ␮mole of glucose equivalents per min ± 4-1 Pond shore 0.007 <0.001 under the conditions tested. Schizophyllan (cosmetic grade) was purchased from 4-2 0.001 ± <0.001 European Technologies, Inc., Denver, CO. Other ␤-glucan substrates (laminarin ± 6-1 Clay soil 0.008 <0.001 from Laminaria digitata, paramylon from Euglena gracilis, curdlan from Agrobac- ␤ 6-2 0.002 ± <0.001 terium sp. (Alcaligenes faecalis), and barley -glucan) were from Sigma-Aldrich, St. 6-3 0.21 ± 0.05 Louis. Commercial cellulases from Aspergillus niger and T. viride (Cellulysin) were ␤ 6-4 0.19 ± 0.01 from Calbiochem (La Jolla, CA). Commercial -glucanases from A. niger, T. lon- gibrachiatum, and Bacillus subtilis, as well as laminarinase from Trichoderma sp., 7-1 Marshland <0.001 were from Sigma–Aldrich. Temperature and pH optima were performed using the 7-2 0.08 ± 0.02 same assay. For studies of pH optima, substrate buffer was titrated to the desired test pH with acetic acid or sodium hydroxide before digestion, then returned to 8-1 Backyard/gardening 0.007 ± <0.001 pH 5 before assays were developed, since the DNS assay is pH sensitive. Enzyme 8-2 area 0.003 ± <0.001 values are the mean of triplicate cultures and are characteristic of repeated exper- 8-3 0.003 ± <0.001 iments. ± 9-1 Laboratory contaminant 0.026 <0.001 Rapid, semi-quantitative ␤-glucanase assays were performed using a solid 9-2 0.032 ± <0.001 medium plate assay. Samples (4 ␮L) were spotted directly onto the surface of freshly 9-3 0.029 ± <0.001 prepared plates containing 0.7% (w/v) Phytagel (Sigma–Aldrich Co., St. Louis) and a 0.05% (w/v) commercial schizophyllan in 50 mM sodium acetate buffer, pH 5.0. Strains were isolated from enrichment cultures containing schizophyllan as a Plates were incubated overnight and stained with an aqueous solution of 1 mg Congo sole carbon source. b red/mL for 45 min, then destained with 1.0 M NaCl for 30 min. Enzyme activity was Pure cultures were grown for 7 days in basal medium containing 1.0% schizo- observed as a clear zone. Laminarinase from Trichoderma sp. was used as a positive phyllan as a sole carbon source. control. Extracellular protein was estimated by the Bradford method [16], with bovine serum albumin as the standard. 2. Methods 2.1. Isolation of novel schizophyllan-degrading strains 2.5. Enzyme purification by fast protein liquid chromatography (FPLC) Soil samples from the area of Peoria, Illinois were serially diluted according to Leathers et al. [10]. Specifically, 2.0 g of soil was diluted into 198 mL of sterile 0.2% Enzyme samples were purified using a fast protein liquid chromatography sys- agar in distilled water (water agar). This was shaken vigorously, and then 10 mL was tem (Biologic Duoflow, Bio-Rad, Hercules, CA). Cell-free culture supernatants were transferred to 90 mL of water agar and mixed well. One milliliter of this suspension applied to a 5.0 mL High Q anion exchange column (Bio-Rad) equilibrated in 50 mM was then transferred into 9 mL of water containing 0.01% Tritron X-100. Aliquots sodium acetate buffer, pH 5.0. Bound protein was eluted with an increasing gradi- (0.1 mL) of these final dilutions were used to inoculate 10 mL enrichment cultures ent of 0.0 to 1.0 M NaCl in the same buffer.
Load more

Recommended publications
  • Handbook of Polysaccharides
    Handbook of Polysaccharides Toolbox for Polymeric sugars Polysaccharide Review I 2 Global Reach witzerland - taad lovakia - Bratislava Synthetic laboratories Synthetic laboratories Large-scale manufacture Large-scale manufacture Distribution hub Distribution hub apan - Tokyo Sales office United Kingdom - Compton India - Chennai Synthetic laboratories Sales office United tates - an iego Distribution hub Distribution hub China - Beijing, inan, uzhou outh Korea - eoul Synthetic laboratories Sales office Large-scale manufacture Distribution hub Table of contents Section Page Section Page 1.0 Introduction 1 6.2 Examples of Secondary & Tertiary Polysaccharide Structures 26 About Biosynth Carbosynth 2 6.2.1 Cellulose 26 Product Portfolio 3 6.2.2 Carrageenan 27 Introduction 4 6.2.3 Alginate 27 2.0 Classification 5 6.2.4 Xanthan Gum 28 3.0 Properties and Applications 7 6.2.5 Pectin 28 4.0 Polysaccharide Isolation and Purification 11 6.2.6 Konjac Glucomannan 28 4.1 Isolation Techniques 12 7.0 Binary & Ternary Interactions Between Polysaccharides 29 4.2 Purification Techniques 12 7.1 Binary Interactions 30 5.0 Polysaccharide Structure Determination 13 7.2 Ternary Interactions 30 5.1 Covalent Primary Structure 13 8.0 Polysaccharide Review 31 5.1.1 Basic Structural Components 13 Introduction 32 5.1.2 Monomeric Structural Units and Substituents 13 8.1 Polysaccharides from Higher Plants 32 5.2 Linkage Positions, Branching & Anomeric Configuration 15 8.1.1 Energy Storage Polysaccharides 32 5.2.1 Methylation Analysis 16 8.1.2 Structural Polysaccharides 33 5.2.2
    [Show full text]
  • Functional Biopolymers Produced by Biochemical Technology Considering Applications in Food Engineering
    Korean J. Chem. Eng., 24(5), 816-826 (2007) JOURNAL REVIEW Functional biopolymers produced by biochemical technology considering applications in food engineering Taous Khan, Joong Kon Park† and Joong-Ho Kwon* Department of Chemical Engineering, *Department of Food Science and Technology, Kyungpook National University, Daegu 702-701, Korea (Received 15 January 2007 • accepted 6 February 2007) Abstract−Polysaccharides are widely used in foods as thickening, gelling, stabilizing, emulsifying, and water-binding agents. The majority of the polysaccharides currently employed in the food industry are derived from plants and sea- weeds. Recently, microbial polysaccharides have emerged as an important set of biothickeners for foods. These bio- polymers have overcome to great extent the flaws associated with the plants and seaweeds polysaccharides. This rela- tively new class of biopolymers has unique rheological properties because of their potential of forming very viscous solutions at low concentrations and pseudoplastic nature. This review deals with the current applications of these micro- bial biopolymers in the food industry with a special focus on the commonly used important exopolysaccharides in this area. Key words: Microbial Exopolysaccharides, Foods Industry, Dextran, Xanthan, Bacterial Cellulose, Gellan, Curdlan, Pullulan INTRODUCTION cally (α- or β-anomers). In conclusion, it is always difficult to con- trol the stereospecificity and the regiospecificity of these products Polysaccharides present a wide array of potential applications,
    [Show full text]
  • Callose-Mediated Resistance to Pathogenic Intruders in Plant Defense-Related Papillae
    This is a repository copy of Callose-mediated resistance to pathogenic intruders in plant defense-related papillae. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/126922/ Version: Published Version Article: Voigt, C.A. orcid.org/0000-0001-7658-7889 (2014) Callose-mediated resistance to pathogenic intruders in plant defense-related papillae. Frontiers in Plant Science, 5. 168. ISSN 1664-462X https://doi.org/10.3389/fpls.2014.00168 Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ PERSPECTIVE ARTICLE published: 28 April 2014 doi: 10.3389/fpls.2014.00168 Callose-mediated resistance to pathogenic intruders in plant defense-related papillae Christian A. Voigt* Phytopathology and Biochemistry, Biocenter Klein Flottbek, University of Hamburg, Hamburg, Germany Edited by: Plants are exposed to a wide range of potential pathogens, which derive from diverse phyla. Vincenzo Lionetti, Sapienza – Therefore, plants have developed successful defense mechanisms during co-evolution with Università di Roma, Italy different pathogens. Besides many specialized defense mechanisms, the plant cell wall Reviewed by: Hans Thordal-Christensen, University represents a first line of defense.
    [Show full text]
  • Gelation and Gel Properties of Gellan Gum and Xyloglucan
    Gelation and Gel Properties of Gellan Gum and Xyloglucan Yoko Nitta 2005 Department of Food and Human Health Sciences Graduate School of Human Life Science Osaka City University ジェランガムとキシログルカンの ゲル化およびゲル特性 大阪市立大学大学院 生活科学研究科 食・健康科学講座 新田 陽子 2005 年 Contents ABBREVIATIONS AND SYMBOLS 1. INTRODUCTION Use of polysaccharides in foods 1 Objective and outline of this thesis 8 Overview of gellan gum 10 Overview of Tamarind seed xyloglucan 14 2. METHODS Rheology 19 Rheological properties of polysaccharide solutions 30 Determination of gel point by rheology 31 Thermal analysis: Differential scanning calorimetry (DSC) 32 Conformational transition of polysaccharides 35 Sol-gel transition of polysaccharide 35 Circular dichroism (CD) 36 Conformational transition of polysaccharides 41 3. GELATION AND GEL PROPERTIES OF GELLAN GUM The helix-coil transition of gellan in gels Introduction 43 Experimental 43 Results and discussion 45 Conclusion 49 The sol-gel transition of gellan affected by thermal history Introduction 50 Experimental 51 Results and discussion 52 Conclusion 59 4. THE EFFECT OF SURFACTANT, SALT, AND SUGAR ON GELATION AND GEL PROPERTIES OF GELLAN GUM The effect of SDS on the gelation of gellan Introduction 61 Experimental 62 Results and discussion 63 Conclusion 69 The effect of the immersion into salt solution on gel properties of gellan Introduction 70 Experimental 71 Results and discussion 71 Conclusion 76 The effect of sugars on gel properties of gellan Introduction 77 Experimental 77 Results and discussion 78 Conclusion 80 Appendix 80 5. GELATION OF XYLOGLUCAN BY ADDITION OF SMALL MOLECULES IN FOODS Gelation of tamarind seed xyloglucan by addition of epigallocatechin gallate Introduction 89 Experimental 91 Results and discussion 92 Conclusion 108 6.
    [Show full text]
  • Regioselective Synthesis of Curdlan Derivatives
    Regioselective Synthesis of Curdlan Derivatives Ruoran Zhang Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Macromolecular Science and Engineering Kevin J. Edgar, Chair S. Richard Turner Judy S. Riffle Maren Roman Abby R. Whittington Nov. 10, 2015 Blacksburg, VA Keywords: curdlan, curdlan derivatives, regioselective synthesis, amination at C-6, tight junction opening, water-soluble Copyright © 2015, Ruoran Zhang Regioselective Synthesis of Curdlan Derivatives Ruoran Zhang Abstract Curdlan, a (1→3) linked linear homopolysaccharide composed of β-D-glucan, is produced by the bacterium Alcaligenes faecalis var. myxogenes. Several strategies to synthesize chemically modified curdlan derivatives have been reported, but there have been few reports of regioselective functionalization at specific positions of the curdlan backbone, especially of aminated curdlan derivatives which have remarkable potential in biomedical and pharmaceutical applications. We demonstrate herein the design, synthesis and characterization of a family of regioselectively aminated curdlan derivatives including 6-deoxy-6-(bromo/azido/amino/amido/ammonium) curdlans starting from 6- bromo/azido-6-deoxycurdlan. A key reaction that enabled the whole synthesis of new curdlan derivatives at C-6 described in this dissertation was the highly selective bromination of curdlan. The resultant 6-bromo-6-deoxycurdlan, prepared with high regioselectivity, was treated with trialkylamines or heterocyclic amines to produce a range of water-soluble curdlan ammonium salts. The bromide was then nucleophilically displaced by sodium azide to produce the versatile precursor 6-azido-6-deoxycurdlan. Its water solubility was enhanced either by the incorporation of hydrophilic trioxadecanoate esters into O-2/4 positions or by the borohydride reduction to afford 6-amino-6-deoxycurdlan.
    [Show full text]
  • Title 21—Food and Drugs
    Title 21—Food and Drugs (This book contains parts 170 to 199) Part CHAPTER I—Food and Drug Administration, Department of Health and Human Services (Continued) ........................... 170 1 VerDate Mar<15>2010 07:41 May 13, 2011 Jkt 223067 PO 00000 Frm 00011 Fmt 8008 Sfmt 8008 Y:\SGML\223067.XXX 223067 wwoods2 on DSK1DXX6B1PROD with CFR VerDate Mar<15>2010 07:41 May 13, 2011 Jkt 223067 PO 00000 Frm 00012 Fmt 8008 Sfmt 8008 Y:\SGML\223067.XXX 223067 wwoods2 on DSK1DXX6B1PROD with CFR CHAPTER I—FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) (Parts 170 to 199) EDITORIAL NOTE: Nomenclature changes to chapter I appear at 59 FR 14366, Mar. 28, 1994, and 69 FR 18803, Apr. 9, 2004. SUBCHAPTER B—FOOD FOR HUMAN CONSUMPTION (CONTINUED) Part Page 170 Food additives......................................................... 5 171 Food additive petitions ........................................... 24 172 Food additives permitted for direct addition to food for human consumption ................................ 30 173 Secondary direct food additives permitted in food for human consumption ....................................... 122 174 Indirect food additives: General .............................. 155 175 Indirect food additives: Adhesives and components of coatings ............................................................ 156 176 Indirect food additives: Paper and paperboard com- ponents ................................................................. 198 177 Indirect food additives: Polymers ..........................
    [Show full text]
  • Polysaccharide Catalogue and Handbook Table of Contents
    Remove dotted spine lines before printing Carbosynth China Head Office Suite 421, Building A2, 218 Xinghu Street, Suzhou Industrial Park, 215123. China +86 512 6260 5585 The Source for Carbohydrates and Nucleosides +86 512 6260 5576 [email protected] Carbosynth Head Office www.carbosynth.cn Axis House, High Street, Compton, Newbury, Berkshire, RG20 6NL. UK Carbosynth Beijing Laboratories +44 1635 578444 CAS - RCEES, 18 Shuangqing Road, +44 1635 579444 Haidian District, Beijing 100085. China [email protected] +86 512 6260 5585 www.carbosynth.com [email protected] www.carbosynth.cn Carbosynth Compton Laboratories 8&9 Old Station Business Park, Compton, Jinan Carbotang Biotech Co. Ltd Berkshire, RG20 6NE. UK Zhangqiu Industrial Development Zone, +44 1635 578444 Jinan, Shandong, 250204. China [email protected] [email protected] www.carbosynth.com www.carbotang.com Carbosynth LLC USA Carbosynth India Suite F, 7887 Dunbrook Road, T - I. Block 2, Jains KK Gardens, San Diego CA 92126. USA Vembuli Amman Kovil Street, West KK Nager, Polysaccharide Catalogue (858)-779-9911 Chennai 600 078. India (888)-681-2933 (Toll Free) +91 44 420 16316 (858)-605-0807 and Handbook [email protected] [email protected] www.carbosynth.com www.carbosynth.com Metal Scavenging Solutions PhosphonicS Limited Axis House, High Street, Compton, Newbury, Berkshire, RG20 6NL. UK +44 1635 577669 +44 1635 579444 Toolbox for Polymeric sugars [email protected] 0800-251302 www.phosphonics.com 服務專線： Inside front cover Inside back cover Carbosynt h P oly sa cc har ides Compton UK Beijing China San Diego USA Jinan China C Suzhou China e llu Chennai India lo s e P ec tin Ex ud at e G .
    [Show full text]
  • Hydrocolloids
    1 1 General Overview of Food Hydrocolloids 1.1 Introduction to the World of Hydrocolloids The term ‘hydrocolloid’ is derived from the Greek hydro ‘water’ and kolla ‘glue’. Hydrocolloids are colloidal substances with an affinity for water. From a chemical point of view, they are macromolecular hydrophilic substances. Some of them are water soluble and form colloidal solutions others are only able to swell in water and can be dispersed by means of shear forces. Hydrocolloids pro- duce viscous solutions, pseudo-gels, or gels in water. The heterogeneous group consists of polysaccharides and proteins. Hydrocolloids are used in technical and regulated applications to thicken and to stabilize formulations. In processed foods, they are ubiquitous – no other group of ingredients contributes more to viscosity, texture, and body like hydro- colloids do. Hydrocolloids are not really emulsifiers because, mostly, they do not have the characteristic linkage of lipophilic and hydrophilic groups in the molecular structure. The molecules are too big and complex in size and therefore are not flexible enough to cover the interfaces being formed during homogenization of oil–water mixtures fast enough to create a long-term stable emulsion with suffi- ciently small droplet diameter. However, these thickeners can stabilize emulsions by increasing the viscosity of the water surface or by interaction with surface- active substances. Some hydrocolloids like gum Arabic or non-ionic products such as methylcellulose (MC), HPMC (hydroxypropylcellulose), or propylene glycol alginate (PGA) reduce the surface tension and exhibit limited emulsifying properties [1]. In accordance to their origin and way of manufacturing, hydrocolloids can be classified in four different groups: 1) hydrocolloids purely isolated from plants (without chemical modification); 2) hydrocolloids obtained by fermentation; 3) plant-derived hydrocolloids that are chemically modified; 4) hydrocolloids from animals.
    [Show full text]